T Cell Mediated Immunity to Influenza
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Transcript of T Cell Mediated Immunity to Influenza
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T cell mediated immunity to influenza:mechanisms of viral control
Nicole L. La Gruta and Stephen J. TurnerDepartment of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of
Melbourne, Parkville, Victoria, 3010, Australia
Infection with influenza A virus (IAV) is a major cause
of worldwide morbidity and mortality. Recent findings
indicate
that
T
cell immunity is
key to
limiting
severity
of disease arising from IAV infection, particularly in
instances where antibody immunity is ineffective. As
such, there is
a
need to
understand
better
the
mecha-
nisms that mediate effective IAV-specific cellular immu-
nity, especially given that T cell immunity must form an
integral part of any vaccine designed to elicit crossreac-tive immunity against existing and new strains of influ-
enza virus. Here, we review the current understanding
of cellular immunity to IAV, highlighting recent findings
that demonstrate important roles for bothCD4+ and CD8+
T cell immunity in protection from IAV-mediated disease.
T cells
and
immunity
to
IAV
infection
Worldwide,
seasonal
IAV
infection
is
a
major
cause
of
morbidity and mortality, estimated to be responsible for
35
million
cases
of
severe
illness
and 250 000500 000
deaths
worldwide
per
annum
(WHO
influenza
centre
web-
site). IAV-specific immunity can be induced byvaccination
that
generates
IAV-specific
antibodies
that
limit
or
preventIAV infection. However, the IAVvaccine needs to be refor-
mulated
on
an
annual
basis
because
influenza
viruses
rapidly
evolve,
with
new
strains
emerging
that
have
lost
or mutated the targets recognized by the preceding anti-
body
response.
Thus,
an
arms
race
ensues
whereby
vac-
cine-induced immune pressures select for new strains that
are
no
longer
recognized
by
vaccine
induced
IAV-specific
antibodies,
necessitating
the
production
of
updated
IAV
vaccines. CD8+ and CD4+ T cells have distinct but impor-
tant
roles
in
the
control,
and
eventual
clearance,
of
influ-
enza virus infection [1]. Upon activation (Box 1), CD4+ T
cells (or helper T cells) are thought to promote effective
immunity
primarily
by
providing
the
necessary
secondary
signals for optimal antibody responses, as well as produc-
ing
antiviral
and
proinflammatory
cytokines
upon
infec-
tion, although recent data indicate their role may extend
beyond
just
cytokine
production
[2]. CD8+ T
cells
are
often
considered
the
hit-men
of
the
immune
system
because
they locate and kill virus-infected cells in the body, thus
limiting
viral
spread
and
contributing
to
the
eventual
clearance
of
infection.
CD8+ T
cells
express
a
range
of
effector genes including granzymes and perforin, which
mediate
their
signature
cytotoxic
capacity.
Given that processing
and presentation
of
viral
peptide
targetson thehostcell surface can only occurafter infection,
unlike
preformed
antibody responses,
pre-existing
T
cell
immunity
cannot prevent
IAV
infectionper se; an issue that
has, in the past, resulted in the dismissal of cellular immu-
nity as a goal of effective vaccination.However, theutility ofcellular immunity
stems
from
the fact that unlike antibody
responses, cellular immunity targets viral proteins that are
more likely to be shared betweendifferentvirus strains and
subtypes [1,3], thereby offering a greater breadth of protec-
tion. Moreover, unlike for
chronic
viruses
such
as
HIV or
hepatitis B virus, wherea primary goal ofvaccination must
be
sterilizing immunity,
for
acute viruses
such
as
IAV,
the
principal
objective
is
the amelioration of
infection-associat-
ed pathology until virus is cleared. Thus, it is widely ac-
knowledged
thata
comprehensive
vaccineagainst
IAV
must
include
the ability to
elicit T
cell
immunity
[4].
Here, we
examine the current state of knowledge regarding IAV-
specific
T
cell immunity
and discuss
how
a
greater under-standing of factors that shape and promote IAV-specific
cellular immunity
will
contribute
to
improved
vaccinestrat-
egies capable of eliciting heterologous immunity.
Targets of the T cell response during influenza infection
The factthatIAV-specific memory T cells cantarget a broad
range of
peptides
derived
from
proteins
that are relatively
conserved betweendifferent
influenzastrains and
subtypes
means that T cell immunity induced by one IAV strain has
the
potential
to
provide immunity
against
distinct IAV
strains
in
the absence
of
neutralizing antibody
(termed
heterologous
immunity).
If
we
are to
take full
advantage
of
IAV-specific
T
cell immunity
via
development
of a
novelT
cellbasedvaccinestrategy, knowing thepreciseIAV peptide
targets recognized by
T
cell immunity
after infection
will
be
key. Lee and colleagues [5] utilized ex vivo stimulation of
human
peripheral
blood
mononuclear cells (PBMCs) with
overlapping
peptides
that spanned the whole
IAV protein
spectrum, to show that individuals who had not been ex-
posed
to
theH5N1
virus
(i.e., seronegativefor
the
virus)
had
both
CD4+ and CD8+ T
cells that could recognize peptides
derived from this highly pathogenic virus. This suggested
thatprevious
infection
by seasonal
influenzacould generate
T cell immunity that was capable of recognizing serological-
lyunrelated IAV
subtypes.
Moreover, theyalsodemonstrat-
ed that the major T cell targets were derived from the IAV
Review
1471-4906/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.06.004
Corresponding author: Turner, S.J. ([email protected]).
Keywords: influenza A virus; CD8+ T cell; CD4+ T cell; immunological memoryMHC
class I MHC class II vaccination.
396 Trends in Immunology, August 2014, Vol. 35, No. 8
http://dx.doi.org/10.1016/j.it.2014.06.004mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.it.2014.06.004http://crossmark.crossref.org/dialog/?doi=10.1016/j.it.2014.06.004&domain=pdf -
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matrix protein (M)1 and nucleoprotein (NP). Studies that
have
alsoused
theapproachof
ex
vivo
stimulation
of
humanPBMCs withoverlapping IAV peptides have since confirmed
thatpeptides
fromtheM1
andNP
are themajor
targetsfor
T
cell immunity [68]. Thus, pre-existing IAV-specific T cell
immunity
inducedbyinfectionwith one
strainmay
have the
capacity (through
cross-reactivity with conserved epitopes
from a limited number ofviral proteins) to limit infection by
different
strains or
subtypes, particularly in
the absence
of
any
neutralizing antibody
responses.
Moreover,novelT
cell
based vaccinestrategieswouldonlyneedto includea limited
number
of
protein
targets
to
ensure
broad-based
immunity
and likelymakevaccineformulation a morestraightforward
exercise than
having
to
use
something
like whole
inacti-
vated IAV.In terms of precise
peptide targets recognized by
human
memory
T
cells, there
remains much
to be
learned. For
example, a robust CD8+ T cell response against a peptide
derived
from the
IAV
matrix
protein
(M1,
residues
5866;
M15866) can be readily detectedwithin HLA-A2+ individuals
[9]. Given the repeateddemonstrationof this responseacross
HLA-A2+ individuals,
it
is
considered a
dominant
response.
Are
other
suchdominant
responses
prevalent
within
individ-
uals with other MHC haplotypes, and what is the full reper-
toire of IAV
peptides eliciting
a
CD8+ T
cell
response? Chen
and
colleagues
addressed these questions by
taking a
sys-
tematic approach
wherein
peripheral humanT
lymphocytes
fromseveralhealthydonors were cocultured with live IAV as
an antigenic stimulus whereby infected
PBMCs
self pre-
sented
IAV antigens
to pre-existing memory T
cells. They
then screened
T
cell
reactivity against individual
influenza
proteins, enabling themtonarrowthe candidate pool, so that
they could
then define the
minimal peptide
targets after in
vitro stimulation with overlapping peptides [10]. As previ-
ouslyreported[11], the dominant CD8+T cell responses from
different
individuals targeted the
conserved
matrix
(M) andnucleoprotein (NP) proteins. Importantly, using this system-
atic approach,
new
peptide targets presented
by an
array
of
distinct
HLA
molecules
were
identified
and
found
to be
at
times more prominent than the benchmark HLA-A2-M1
epitope. Interestingly, it
was noted
that
at
least three
of
the newly identified IAV-peptide targets identified were
longer than
typical
MHC class I binding peptides, and
hence
would not
have been
identified using
established
epitope
prediction algorithms [10].
These
results
suggest
that a
more
systematic
and direct
approach,
such
as
that outlined in
the study
by
Chen and
colleagues [10], is needed if peptide targets for a range of
diverse
MHC
alleles
are to
be
identified
for
possible inclu-
sion in T cellbased vaccine approaches.Although the use ofspecificpeptides
in
vaccinesis
a
direct
way
of targetingT
cell
response,theapplication of targetedpeptide based vaccines
will
likely be
limited by the fact that potential
epitope
targetswill
be
missed,
as
well
as
by
thedifficulty
of
ensuring
adequate coverage across numerous HLA subtypes. More-
over,
there
is
an
increasing appreciation
that IAV-specific
T
cell immunity
may
in
fact drive
immune
escape
in
targeted
T
cell epitopes (Box 2). Alternatively, vaccine strategies that
incorporate
whole
protein
antigens, rather than peptides,
would ensure
adequate
antigenic coverage across different
HLA types. Aside from ensuring broad T cell immunity,
another
advantage
of
whole
protein
vaccinationagainst
IAV
would
be
thepotential
to
maximize
fully
humoral
responseseither against conserved proteins,
such
as
MP
or
NP
[12],
or
conserved protein structures such as the stem region of the
hemagglutinin
(HA)
[13];
bothof
which
have
shown
promise
in
protection
from heterologous
challenge.
It is important to note that whole proteinvaccine strat-
egies do
not
circumvent
the
need
for
high-resolution
epi-
tope
identification.
A
major
driver
is
the
increasing
need
to
be able to track antigen-specific T cell responses after
vaccination
by
use
of
soluble
pMHC
tetramer
reagents
[14]. The identification of new IAV-specific pMHC com-
plexes, combined with recent advances in flow cytometric
techniques that enable multiple specificities/parameters to
be
measured
from
a
single
blood
sample
[15,16], means
we
are
potentially
at
the
beginning
of
an
era
that
will
provide
an unprecedented level of information about the kinetics,
function
and
persistence
of
IAV-specific
T
cell
immunity.
Role of T cell immunity against IAV infection: lessons
from humans
Although
IAV-specific
CD4+ and
CD8+ T
cells
are
readily
identifiable in humans [5,1720], their precise role in
controlling
IAV
infection
is
unclear.
A retrospective
analy-
sis
demonstrated
that
prior
symptomatic
A(H1N1)
infec-
tion
was
associated
with
increased
protection
from
the
1957 A(H2N2) pandemic virus in adults but not children,
suggesting
an
accumulation
of
heterologous
immunity
Box 1. Viral escape from cellular immunity: a paradox of
acute
infections
DCs are a specialized subset of antigen-presenting cells that are key
for alerting the host to infection and initiating T cell responses. DCs
exist as twogeneral populations; those located in peripheral tissues
and those located within secondary lymphoid tissues such as the
lymph nodes. At least within the murine system, DCs located within
these locations can be further divided into distinct subsets, with
each reported to have distinct roles in antigen presentation andpriming of T cell responses [58]. DCs within the lymph nodes can be
broadly separated into theCD11b+ CD8a or CD11b CD8a+ subsets,
with the CD8a+ DCs being most efficient at presenting influenza
antigens and activating nave, virus-specific T cells after infection
[59]. Within peripheral t issues, DCs can be divided into CD103+
CD11b or CD103 CD11b+ DCs, with the CD103+ DCs capable of
migrating most efficiently to the draining lymph nodes after IAV
infection [60]. Importantly, in the context of IAV infection, both lung-
derived and lymph-node-derived DCs appear to play roles in the
induction of T cell immunity to influenza [58,61]. For CD8+ T cell
responses, both the CD8a+ CD11b (LN) and CD103+ CD11b (lung)
DC subsets are important for priming [6062]. Although priming of
CD8+ T cell responses is essentially limited to CD8a+ (LN) and/or
CD103+ (lung) derived DCs, a broader range of DC subsets are
capable of presenting antigen to CD4+ T cells [63]. However, in the
context of IAV infection, the migratory CD11b
CD103+
DCs derivedfrom the lung are capable of activating nave CD4
+ T cell responses
[62,64]. Although activated B cells can also present antigen to CD4+
T cells, the key purpose of this TB interaction is the promotion of
effective antibody responses, rather than initial priming of nave
CD4+ T cell responses. Finally, although there is little information
regarding the role of human DC subsets in priming IAV-specific T
cell responses, subsets analogous to the mouse CD8a and CD103+
DC subsets have been identified in humans and are most efficient at
priming nave CD8+ T cell responses [65,66]. Thus, determining
whether these same DC subsets are key players in the initiation of
IAV-specific T cell responses in humans after infection/vaccination is
of great interest and relevance to T cell based vaccine design.
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with age [21]. Although the mechanism is unknown, the
fact
that
protection
was
mediated
in
the
absence
of
any
crossreactive
antibody
responses
(because
it
was
a
pan-demic
event),
strongly
suggests
a
key
role
for
T
cell
medi-
ated protection [21].
The
earliestindirectevidence
in
humans
thatCD8+T
cell
immunity
is
important for
protection against
influenza-
mediated illness came from a challenge study in which
volunteerswere
intranasally
infected
with
a
live, attenuat-
ed
IAV,
and
viral
shedding
was
measured
in
clinicalsamples
[20]. Decreased viral shedding was associated with a con-
comitant increase in
IAV-specific
CD8+ T
cell
responses in
volunteers who
lacked neutralizing, strain-specific
antibo-
dies.
These
findings
implied that IAV-specific
CD8+ T
cell
responses could effectively limit primary IAV infection.
The
recent
2009
H1N1
pandemic
(2009
pdmH1N1)
provided
a
unique
opportunity
to
determine
whether
pre-existing CD8+ T cell immunity provides protection
from
heterologous
IAV
infection.
In
one
particular
study
[7],
a
cohort
of
individuals
that
lacked
pre-existing
anti-
bodies to the 2009 H1N1 IAV pandemic were followed
during
pandemic
cycles
to
determine
whether
pre-existing
circulating
memory
T
cell
populations
correlated
with
less
severe disease outcomes after 2009 pdmH1N1 infection.
Individuals who developed mild or no symptoms after 2009
pdmH1N1 influenza infection were found to have higher
circulating
levels
of
pre-existing
IAV-specific
CD8+ effector
memory T cells (defined by CD45RA+ CCR7 expression).
Functionally,
these
effector
memory
CD8+ T
cells
exhibited
the
capacity
to
produce
interferon
(IFN)-g
and
were
capa-
ble
of
direct
cytotoxicity
against
infected
target
cells.
In-
terestingly,
there
was
no
significant
correlation
between
symptom severity (symptoms noted were runny nose, fe-
ver,
and
sore
throat)
and
the
presence
of
pre-existing
IAV-
specific
CD4+ T
cells.
Thus,
in
the
setting
of
natural
infec-
tion, when antibody immunity is lacking, elevated influen-
za-specific
CD8
+
T
cell
immunity
appears
to
help
limit
bothdisease symptoms and the spread of the virus.
As
noted
earlier,
both
CD4+ and
CD8+ T cell
responses
can
target
relatively
conserved
internal
influenza
proteins,
implying that CD4+ T cells may have the potential to
provide
IAV-specific
heterologous
immunity
[5]. To
test
this directly, Wilkinson and colleagues [8] challenged
volunteers
with
either
a
wild
type
H1N1
or
a
H3N2
sea-
sonal
IAV
strain,
and
then
measured
clinical
symptoms
and viral shedding over the course of infection. All volun-
teers
were
seronegative
for
their
respective
challenge
strain,
therefore,
the
levels
of
pre-existing
memory
T
cell
responses were measured to determine their relation with
disease
progression.
Although
no
significant
correlation
was found between symptom severity and presence ofpre-existing
IAV-specific
CD4+ T
cells
[7],
Wilkinson
et al. found a significant inverse correlation between dis-
ease
severity
and
pre-existing
levels
of
circulating
IAV-
specific
CD4+ cells.
It
is
not
clear
why
these
two
recently
published IAV challenge studies came to different conclu-
sions
about
the
respective
roles
of IAV-specific
CD8+ and
CD4+ T
cells.
It
might
be
the
fact
that
Sridhar
and
collea-
gues examined a natural experiment, whereas Wilkinson
et al. analyzed heterologous IAV-specific T cell responses in
the context
of
an
experimental
challenge
of human
volun-
teers. Alternatively, analysis of T cell populations found
within
the
respiratory
tract
during
IAV
infection
may
provide
stronger
correlates
of
immunity.
Although
morestudies
like
these
are
needed
before
any
definitive
answer,
these findings nevertheless provide strong impetus for
further
developing
an
understanding
of both
CD8+ and
CD4+ T
cell
effector
functions
and
their
role
in
IAV
control.
Mechanisms of CD8+ T cell dependent control of IAV
infection
Signaturevirus-specific CD8+T cell effectorfunctionsinclude
the
ability to
produce
a
variety
of cytotoxicmolecules
such
as
perforin (Pfp) and granzymes (gzm), as well as being able to
secrete a variety of potent inflammatory cytokines such as
tumor necrosis factor (TNF)a and IFN-g (Figure 1). It is
natural
to expect that
perhaps
many,
if
not
all,
of
these
effector
functions
contribute to the
limiting
and
eventual
clearance of IAV infection. Pfp-deficient mice display an
impaired
capacity to clear
IAV
infection,
suggesting
that
Pfp-dependentcytotoxicityplays
a
majorrole
[22]. Unexpect-
edly, mice deficient in the major granzyme proteins,A and B,
do not
show heightened susceptibility
and
can
control IAV
infection
as
effectively
as
wild
type mice
[23]. This
suggests
that other cell death pathways, such as FasFas ligand
interactions
mediated by
activated T
cells
[22]
or
other
death-domain-containing
proteins
such
as
TNF-related apo-
ptosis inducing
ligand
(TRAIL) [24], may
havea
role. Amore
intriguing possibility is that other granzymes, such as grzK,
can compensate for
the
loss of grzA
and
B
and
contribute to
Box 2. Viral escape from cellular immunity: a paradox of
acute
infections
The acute nature of IAV infection is not typically considered to be a
strong driver of mutational escape within targeted T cell epitopes
because the duration of virus infection and the subsequent immune
response is thought to be insufficient for the outgrowth of viral
escape mutants. However, recent studies that examined the
evolution of amino acid sequences within the relatively conserved
NP from an array of different IAV isolates taken over the past 40years reported a high frequency of mutation. It was subsequently
shown that these amino acid changes within known NP-derived
CD8+ T cell epitopes resulted in loss of CD8+ T cell recognition of
IAV-infected cells [67]. Importantly, amino acid variation is not
limited to a single CD8+ T cell epitope, with variations identified
within a number of other known NP-derived CD8+ T cell peptides
that bind numerous different human MHC class molecules [6770].
In a C57BL/6J mouse model of IAV infection, it was recently
demonstrated that viral variants containing mutations at the MHC
class I position 5 anchor residue of the DbNP366 epitope emerged
coincidently with the peak of the DbNP366-specific CD8+ T cell
response. When these viral variants were used to inoculate MHC-
mismatched Balb/c mice (and were thereby relieved of T cell
immune pressure) the variant IAVs reverted back to the wild type
NP sequence. These data demonstrate that even a primary CD8+
T cell response to IAV has the capacity to exert sufficient immunepressure to select escape variants. Although not as extensively
studied, there is also some evidence that mutationwithinCD4+ T cell
epitopes can result in escape from IAV-specific CD4+ T cell
recognition. Given that T cell epitopes tend to be more conserved
between different IAV strains than those recognized by antibody
immunity, these data emphasize the need to
select carefully
potential antigens for inclusion in any future vaccine strategy, so
that although a range of T cell reactivates are included, it might be
necessary to exclude antigens that, from an evolutionary perspec-
tive, appear to be targets of immune T cell selection.
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limiting
and
eventual
control
IAVinfection.
grzK
is
expressed
at high frequency by
both
mouse
and
human
virus-specific
CD8+ T cells [2528], and is also expressed at high levels in
IAV-specific
CD8+ T
cells
in grzA/B-deficient
mice
[23]. It is
possible that
perhaps
grzK
is
the
key cytolyticmolecule
that
mediates CD8+ T cell killing of virus-infected cells, and it
remains to be
determined
whether
grzK
can
compensate
for
the loss of
grzA
or
B.
IAV-specific CD8+ T cells can simultaneously produce a
variety
of
proinflammatory
cytokines
in
response
to
anti-
gen activation
[29]. Effector
CD8+ T
cells
isolated
from
bronchoalveolar
lavage
exhibit
a
heightened
functional
capacity, particularly in terms of proinflammatory cyto-
kine
production
[25,29]. Secretion
of
these
mediators
preferentially
occurs
at
sites
of
active
infection
where
there
is
increased
presentation
of
viral
determinants
and
a
pre-
existing inflammatory environment as a consequence of
innate
inflammatory
mediators
[30]. At
least
in
mouse
experimental
systems,
lung-resident
memory
T
cells
can
persist long term after IAV infection, where they are
thought
to
constitute
a
frontline
defense
against
secondary
challenge
[31]. Following
secondary
IAV
challenge,
the
lung-resident memory T cells are supplemented by chemo-
kine
CC
receptor
(CCR)5-dependent
recruitment
of
circu-
lating
memory
CD8+ T
cell
to
the
infected
lung
[32]. Both
the
resident
memory
and
newly
recruited
IAV-specific
CD8+ T cells can immediately secrete IFN-g upon antigen
recognition
and
contribute
to
early
virus
elimination
[33].
(A)
Lung airways
Cytokine
TH
1 TH
17
Cytotoxic
FasL
Trail
MHCI
MHCII
IL-10
Perforin
Gzm A, B K
Perforin
Gzm A, B
IL-2
IFN
IFN IL-17A
IL-6
IL-21
TNF
TNF-
IL1-
CXCL9
CCL2
Lung parenchyma
CCR5+CCR5+
Blood vessels
(B)
(C)
(D)
TRENDS in Immunology
Figure 1.
T cell effector mechanisms in the IAV infected lung. (A) Recently activated IAV-specific CD8+ and CD4+ T cells are recruited to infected lung tissue in a CCR5-
dependent manner.During a secondary response, recruitment of newly activated memory T cells supplements lung-resident memory T cells that remainedin the lungafter
resolutionof a primary infection. (B) IAV-specificCD8+ T cells recognize IAV-infected lungepithelial cells presentingMHCclass I molecules presenting IAV-derivedpeptides.
Upon T cell receptor recognition, effector CD8+ T cells contribute to viral control and elimination via a combination of mechanisms including: (i) delivery of cytotoxic
moleucles such as perforin and granzymes; (ii) secretion of proinflammatory cytokines such as IFN-g and TNF-a; and (iii) expression of death domain receptors FasL and
TRAIL that can initiatecell death afterbinding to their respective ligands.At later stages of infection, IAV-specificCD8+ T cells canalsoexpress IL-10 as a wayof helping limit
T cell dependent immunopathology in the lung. (C) IAV-specific effector CD4+ T cells contribute to viral control and elimination via secretion of either TH1 or TH17
proinflammatory cytokines. UpregulationofMHCII on inflammed epithelial cells means thatCD4+ T cells candirectly recognize infected cells,and there is a
suggestionthat
lungeffectorCD4+ T cells mayalsomediatedirect cellcytotoxicity viadeliveryof perforinand granzymes. (D)ActivatedeffectorCD4+ T cells canalso trigger thesecretion of
innate cytokines such as IL1-b, CXCL9, and CCL2 helping contribute to the proinflammatory response in the infected lung. The cellular source of these cytokines is not
known are likely to be lung residentmacrophages. Abbreviations: CCL2, chemokine CC ligand 2; CCR5, chemokine CC receptor 5; CXCL9, chemokine CXC ligand 9; Gzm,
granzyme; IAV, influenza A virus; FasL, Fas ligand; IFN, interferon; IL, interleukin; TH, T helper; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand.
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The
recruitment
to
the
murine
lung
of
these
highly
active
memory
CD8+ T
cells
expressing
IFN-g
has
been
shown
to
be
key
for
protection
against
influenza
infection
[34].
The enhanced effector potential of lung localized CD8+T
cell
effectors
also
increases
the
risk
of
damage
to
lung
tissue
due
to
excessive
inflammation
(reviewed
in [35]).
Thus, to mitigate damage to the sensitive lung tissue by
potent
T
cell
effector
functions,
a
balance
must
be
struckbetween ensuring effective antiviral potency while not
causing
immunopathology.
It
is
intriguing
that
IAV-spe-
cific
CD8+ effector
T
cells
isolated
from
infected
mouse
lungs are capable of producing interleukin (IL)-10; a potent
negative
regulator
of
inflammation
[36]. The
ability
of
effector CD8+ T cells to produce IL-10 is dependent on
migration
into
the
inflamed
lung
[37], suggesting
that
there
are
signals
specific
to
the
infected
lung
microenvi-
ronment that trigger regulatory functions in otherwise
proinflammatory
IAV-specific
CD8+ T
cells.
In
this
way,
the immune
response
can
balance
the
need
for
inflamma-
tion required to clear IAV infection, with the need to limit
tissue
injury
by
the
inflammatory
response.
Mechanisms
of
CD4+ T
cell
control
of
viral
infection
Classically, activated CD4+ T cells are considered to be key
for
promotion
of
effective
antibody
responses
via
support
of
germinal
center
formation
that
results
in
affinity
matura-
tion and isotype switching [3841]. This occurs through the
provision
of
key
co-stimulatory
signals
such
as
inducible
T
cell
co-stimulator
(ICOS),
and
the
production
of
cytokines
such as IL-21 [40,41]. The antibody response to IAV infec-
tion
is
critical
for
protection
[42]; lack
of
neutralizing
antibody
levels
in
the
population
is
a
key
factor
controlling
the emergence of IAV pandemics [43].
Thefinding
that
memory
CD4+ T
cell
responses
contrib-
ute
to
heterosubtypic
immunity
against
a
potential
pan-demic
IAV
[8]
suggests
that
memory
CD4+ T
cells
play
a
key role in the control of IAV infection, but the mechanisms
involved
are
not
clear.
Adoptive
transfer
of
a
large
number
ofex vivo isolated memory IAV-specific CD4+ T cells into a
mouse model of infection augmented both IAV-specific
CD8+ and
B
cell
responses
against
primary
infection
[44]. A
more
detailed
analysis
of
how
memory
CD4+ T
cells
can promote primary IAV-specific B cell response has
shown
that
establishment
of
NP-specific
memory
CD4+
T cells by peptide vaccination of mice promoted robust
germinal center formation and a more rapid primary
NP-specific antibody response after IAV infection, com-
pared
to
unvaccinated
mice
[45]. Strikingly,
memory
NP-specific
CD4+ T
cells
did
not
promote
antibody
responses to otherviral proteins, including the HA protein,
the
major
target
of
the
antibody
response.
This
suggests
that
both
the
antibody
and
T
cell
responses
are
linked
to
the sameviral target; likely as a consequence of the ability
of B
cells
to
process
and
present
CD4+ T
cell
epitopes
from
antigen
captured
and
internalized
via
surface
immuno-
globulin receptors.
Mouse
models
of
IAV
infection
provide
a
tool
whereby
CD4+ T
cell
effector
mechanisms
can
be
delineated
more
precisely
(reviewed
in [46]). For
example,
recent
studies
have utilized adoptive transfer of T cell receptor (TCR)
transgenic
CD4+ T
cells
specific
for
an
epitope
of
the
HA
protein
of
an
H1N1
IAV [A/PR8/34
(HNT)]
to
determine
the contribution
of
CD4+ T
cells
to
protection
from
IAV
infection
[44].
Initial experiments
demonstrated that
adoptive transfer of CD4+ HNT T cells that were differen-
tiated in vitro into proinflammatory T helper (TH)1 or
TH17 lineages (Figure 1) were more capable of mediating
clearance and protection from IAV infection, compared to
uncommitted
(TH
0) or
anti-inflammatory (TH
2) CD4
+
effectors [44]. This control was partly via augmentation
of endogenous IAV-specific
CD8+ T
cell
and B cell
responses
and
partly
via triggering expression
of innate
cytokines such as IL-1b, IL-6, chemokine CXC ligand
(CXCL)9 and chemokine CC
ligand (CCL)2, particularly
within the infected lung[47].
The
demonstration
that
at
least
in
murine
models,
influenza
infection
can
induce in vivo MHC class II expres-
sion on lung epithelial cells [48] highlights the possibility
that
CD4+ T
cells
could
have
a
role
in
control
of
IAV
infection
by
directly
recognizing
and
eliminating
virus-
infected targets. In fact, protection from IAV infection
conferred
by
adoptive
transfer
of
memory
HNT
CD4+ T
cells into immunodeficient mice was abrogated when thesecells
were
deficient
in
either
IFN-g
or
perforin
[44]. Hence,
aside from providing help to B cells and CD8+ T cells, IAV-
specific
CD4+ T
cells
have
the
capacity
to
target
directly
IAV-infected
cells,
thereby
contributing
to
the
control
and
elimination of IAV infection. Such unconventional mecha-
nisms
of
T
cell
action
must
be
appreciated
for
the
strategic
design
of
vaccines
aiming
to
elicit
effective
cellular
immu-
nity.
CD4+ T cell regulation of IAV-specific CD8+ T cell
responses
The precise
role
of
CD4+ T cells
in
promoting
and
regulat-
ing
CD8+
T
cell
responses
induced
by
IAV
infection
was,until
recently,
enigmatic.
This
is
partly
because
inmice,
an
effective primary CD8+ T cell response to IAV can be
induced
independently
of
CD4+ T
cells
[49]. In
this
case,
direct
activation
of
dendritic
cells
(DCs)
via
the
engage-
ment of Toll-like receptors (TLRs) by IAV circumvents the
need
for
CD4+ TH-dependent CD40 ligand (CD40L) licens-
ing of DCs
to
promote
primary
virus-specific
CD8+ T
cell
responses [50].
However, memory
CD8+ T
cells
that
are
primed
in
the
absence of CD4+T cells are reduced in number and show an
inability to response to secondary infection, compared to
memory CD8+ T cells primed in the presence of CD4+ T
cells
[49]. So,
although
dispensable
for
primary
activation
and expansion,
CD4+ T
cell
help
is
crucial
(during
the
initial priming phase) for programming optimal IAV-spe-
cific
CD8+ T
cell
memory.
The
role
of
CD4+ T
cell
help
in
this
case
is
the
provision
of
co-stimulatory
signals
via
CD40LCD40-dependent interactions with DCs that lead
to optimal
priming
of
the
IAV-specific
CD8+ T
cell
re-
sponse.
These
signals
received
from
licensed
DCs
then
ensure the responding CD8+ T cells are capable of auto-
crine
IL-2
production
[51], which
is
crucial
for
their
surviv-
al into
memory.
What
remains
unclear
are
the
precise
signals
provided
by
CD4-dependent
licensing
of
DCs
that
ensure responding CD8+ T cells can establish effective
memory
populations.
Uncovering
these
mechanisms
would
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provide
information
crucial
to
the
design
of
any
CD8+ T
cell
based
vaccine
strategy
to
promote
optimal
memory
formation.
CD4+ T regulatory (Treg) cells have been shown to limit
effector
CD8+ T
cell
differentiation
in
response
to
virus
infection
and
immunization
[5254],
and
they
have
the
capacity to suppress potently primary IAV-specific CD8+ T
cell
responses
after
infection
of
mice
[55,56]. So,
how
is
aneffective primary CD8+ T response sustained in the face of
CD4+ Treg
cell
mediated
suppression?
Recent
work
from
Randall
and
colleagues
suggests
that
activation
of
CD40L+
CD4+ THcells early after IAV infection is key to ensuring
appropriate
DC
activation
that
serves
to
limit
the
expan-
sion and activation of Treg cells [55], which in turn limits
Treg
cell
suppression
of
the
primary
CD8+ T
cell
response
during
the
early
phases
of
infection.
As
the
infection
is
cleared and antigen becomes limiting, Treg cells begin to
exert
their
suppressive
effects
and
effectively
promote
the
tapering
of
the
effector
CD8+ T
cell
response
during
the
contraction phase [55]. In this way, Treg cells may limit
potential
damage
caused
by
a
prolonged
CD8+ T
cell
re-
sponse at later stages of infection. This mechanism is onlyrecently
described
and
raises
several
questions.
For
exam-
ple, is the induction of Treg cells diminished in the case of
highly
pathogenic
IAV
infection,
where
increased
immu-
nopathology
is
associated
with
highly
pathogenic
H5N1
or
the recent H7N9 infection of humans?
The
overall
picture
is
that
is
that
all
arms
of
the
adaptive
response
have
a
role
to
play
in
the
control
of
IAV infection. B cell/antibody-mediated immunity plays
the
major
role
when
it
comes
to
preventing
infection
with
antigenically
matched
strains.
In
cases
where
antibody
reactivity is limiting or absent, there is mounting evidence
that
both
CD8+ and
CD4+ T
cells
have
key
roles
in
limiting
IAV
infection,
particularly
in
the
case
of
heterologous
IAVchallenge.
What
has
begun
to
emerge,
yet
remains
incom-
pletely understood, is the range and redundancy of mech-
anisms
utilized
by T
cells
in
both
controlling
infection
and
limiting
immunopathology,
as
well
as
the
precise
interac-
tions between the adaptive immune cell populations. When
considering
the
development
of
new
vaccines
for
engaging
heterologous
T
cell
immunity,
it
is
essential
that
these
features of T cell activity be understood to ensure estab-
lishment
of
an
effective
memory
T
cell
population.
Concluding remarks
Although there has long been acknowledgement that cel-
lular
immunity
to
IAV
plays
a
role
in
protection
from
infection,
it
is
only
with
recent
advances
in
the
identifica-
tion and isolation of IAV-specific T cells that this has been
accepted
as
an
important
immunological
correlate
of
pro-
tection
from
IAV
infection.
Given
the
extremely
high
mu-
tation rate of the influenza proteins (NA and HA) typically
targeted
by
antibodies,
it
is
becoming
clear
that
protection
from
IAV
infection,
and
a
broader
range
of
infections
such
as HIV, hepatitis Cvirus, and malaria, will requirevaccine
strategies
that
induce
robust
and
long-lived
T
cell
responses
[57]. The
improved
capacity
to
enumerate
and
isolate
IAV-specific
T
cell
responses
has
also
allowed
great-
er insight into the dynamics, location, gene expression, and
genomic
organization
of
IAV-specific
T
cell
immunity
at
distinct
stages
of
T
cell
immune
response
[57]. Although
such
analyses
are
greatly
enhancing
our
understanding
of
both
molecular
regulation
and
immune
mechanisms,
the
practical challenge of how best to manipulate both CD4+
and
CD8+ T
cell
responses,
particularly
via
vaccination,
to
achieve
a
measure
of
long-term,
if
partial,
heterosubtypic
protection is still ahead of us.
Acknowledgments
This work is supported by an Australian Research Council Future
Fellowship (awarded to S.J.T.); a Sylvia and Charles Viertal Senior
ResearchFellowship (awarded to N.L.L.); Australian NationalHealth and
Medical Research Council (NHMRC) program grant 5671222 (awarded to
S.J.T.) and NHMRC project grant AI1046333 (awarded to N.L.L.).
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8/11/2019 T Cell Mediated Immunity to Influenza
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